cDNA sequences too similar to be real? - (Aug/16/2006 )

Hi,

I am wondering if anyone has an idea about this situation.

I am trying to sequence the same gene from several different organisms, that have not been previously known to have this gene. I made universal primers, did the PCRs, got products, and then cloned and sequenced them.

The problem is that the sequences came back much more similar to one another than I would have expected.

I am having doubts about whether there could be contamination of the RNA or of one of the PCR products into the others. BUT, on the other hand, the sequences were 2-3% different (in 500 bp).

Is it possible that cloning or another step might introduce small errors into the sequences, if I have been just sequencing the gene from one of the organisms, due to contamination?

Thanks

-smurray-

QUOTE (smurray @ Aug 16 2006, 04:52 PM)

Do a negative control which has no template to confirm if you have contamination of PCR products.

Use a proofreading polymerase to be more confident that mutations are not being introduced by the polymerase

Have these organisms had their genome sequenced?

-JPStewart-

Hi there,

thanks for your suggestions.

I always have a negative (ddH20) control in the PCR and its always blank.

BUT, I am now thinking that its possible that there is contamination from one of my PCR product in one of the RT buffers, because I don't always use a -RT control that I carry through into the PCR.

Unfortunately, the genome of these organisms have not been sequenced, so its hard to know how similar they might be to others.

I think I will order new RT buffers and try from the RNA again....

cheers

QUOTE (smurray @ Aug 17 2006, 11:52 AM)

-smurray-